Tubing fluid containing an amino acid



United States Patent M 3,345,306 TUBING FLUID CONTAINHQG AN AMINO ACID Alfred Bloch, Highland Park, N.J., assignor to Ethicon, Inc., a corporation of New Jersey No Drawing. Filed Feb. 12, 1964, Ser. No. 344,175 18 Claims. (Cl. 252-403) ABSTRACT OF THE DISCLOSURE A tubing fluid for absorbable sutures has dissolved therein from about 0.01 percent to about 0.2 percent of a monocarboxylic amino acid. The presence of the amino acid minimizes changes that occur in the in vivo absorption characteristics of the suture upon storage.

The present invention relates to a system of tubing surgical materials, and more specifically to a tubing fluid adapted to be used in the packaging and storage of absorbable collagen sutures.

In the manufacture of sutures the catgut strands are often treated with formaldehyde to tan and harden the strand so that it may be polished to a circular cross-section. In addition, catgut is often treated with formaldehyde for the purpose of increasing its resistance against digestive forces in the human patient and to prolong the time during which it is strong enough to hold wound edges together. This is particularly true for absorbable collagen sutures manufactured from tendon or other collagen sources by extrusion and reconstitution of collagen dispersions.

While the amount of formaldehyde that combines with the catgut during the tanning or hardening step can be regulated by such factors as formaldehyde concentration and the pH of the tanning bath, the control of this tanning process is limited. The limitation is caused by the fact that the collagen used is a product of nature which shows chemical and morphological diiferences that are dependent upon the animal source. There is also some evidence that the reaction by the collagen and formaldehyde proceeds slowly after the catgut strand has been packaged in tubing fluid and sterilized. Thus catgut which has been stored for many months or years prior to use may be more highly tanned and may absorb more slowly than freshly manufactured material.

It is common practice as well-known to those skilled in this art to package catgut in a tubing fluid that comprises a low molecular weight alcohol, such as ethanolor isopropanol, and a small amount of water, i.e., 4-25 This mixture of alcohol and water controls the swelling and pliability of the collagen strand Which otherwise would become hard and brittle during storage. Again there is evidence that during prolonged storage traces of the alcohol present in the tubing fluid may be oxidized to an aldehyde which further tans the collagen and results in an increased absorption time (delayed absorption in the human body). The tubing fluid of the present invention regulates and controls the aldehyde content of collagen sutures and diminishes the variability in absorption time.

The tubing fluid of the present invention comprises a solution of a monocarboxylic amino acid in a solvent containing from 75% to 96% by weight of alcohol having no less than 2 and no more than.3 carbon atoms, and 25% to 4% by weight of water.

Suitableamino 'acids for use in the present invention are alanine, arginine, cysteine, glycine, histidine, hydroxy proline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine, as well as the acid salts of these amino acids that are soluble in aqueous alcohol solutions. The dicarboxylic amino acid,

Patented Oct. 3, 1067 such as aspartic acid and glutamic acid are not effective for the purpose of the present invention.

Suitable alcohol for use in the tubing fluid of the present invention are ethanol, propanol, and isopropanol. The amount of alcohol may vary from to 95% by weight of the tubing fluid. If the alcohol employed is isopropanol, the amount of alcohol may vary from about 75% to 90% by weight. If the alcohol employed is ethanol, the amount of alcohol may vary from about to about 96% by weight of the tubing fluid.

The amount of amino acid in solution may vary from as little as 0.0l% up to the maximum solubility of the amino acid or its salt in the aqueous alcohol tubing fluid. While the present invention is not to be limited by any particular theory of operation, it is believed that the amino acid in the tubing fluid reacts with the unbound formaldehyde that remains in the collagen suture and a'ldehydes that may be formed during storage. As little as 0.01% of a low molecular weight amino acid, such as glycine is effective for this purpose. The solubility of specific amino acids in a tubing fluid consisting of isopropanol and 10% water at 40 F. was found to be 0.15% for lysine, 0.2% for arginine, and 0.05% for glycine. It is desirable that the concentration of amino acid be as great as possible without turbidity or precipitation. Stable solutions containing more of the amino acid may be obtained by increasing the percentage of water, replacing isopropanol with ethanol, or converting the amino acid to an acid salt, i.e. by treating it with hydrochloric acid.

The invention will be better understood by reference to the following examples in which all amounts are expressed in parts by weight.

EXAMPLE I In order to demonstrate the effect of acetaldehyde upon the digestibility of surgical catgut, small amounts of this aldehyde, namely 40 p.p.m. and 100 p.p.m., respectively, are added to a tubing fluid consisting of 90% isopropanol and 10% water. The formaldehyde tanned sutures and ten chrome tanned sutures, each 20 inches in length are placed in individual aluminum foil suture packets. To each packet is added 2.5 ml. of the tubing fluid containing 40 p.p.m. acetaldehyde. Each packet is then sealed and sterilized by irradiation with ,B-rays.

Ten formaldehyde tanned sutures and ten chrome tanned sutures each 20 inches in length are placed in individual foil suture packets to which is added 2.5 ml. of the tubing fluid containing 100 p.p.m. acetaldehyde. These packets are sealed and sterilized by irradiation with ,B-rays.

Twenty control samples are prepared in the identical manner except that no acetaldehyde is added to the tubing fluid. After storage for 10 days at room temperature, the 60 suture samples were removed from their packages and tested for their resistance against digestion by the proteolytic enzyme trypsin.

In this test, the catgut sutures are cut to pieces of 1 inch and immersed in a solution of 2% trypsin in phosphate buffer at pH 7.4. They are incubated in this buffered trypsin solution at 100 F. and observed several times daily. The control samples of plain (formaldehyde tanned) and chromic catgut sutures are digested in on and two days respectively. The samples that had been packaged in tubing fluid containing 40 p.p.m. and 100 p.p.m. acetaldehyde show no proteolytic attack after two days; and are not completely hydrolyzed after nine days incubation. These observations demonstrate the effect which small amounts of acetaldehyde can exercise on the digestibility of catgut.

A visual sign of the reaction of the acetaldehyde present in the tubing fluid with the sature is observed in form of an orange-brown discoloration of the plain catgut samples. This discoloration cannot be leached out and may be related to the well-known Maillard reaction, observed to occur when proteins are treated With reducing sugars. In the Maillard reaction, the reactive part of the sugar is believed to be an aldehyde group.

In another experiment a similar tanning reaction was observed to occur after ten weeks storage in tubing fluid containing only p.p.m. acetaldehyde.

EXAMPLE II TABLE 1 Digestion time Tubing fluid in days Neutral TF, control Neutral TF plus 100 p.p.m. acetaldehyde l2. Neutral TF plus 100 p.p.m. acetaldehyde plus 0.15% lysine Neutral TF plus 100 p.p.m. acetaldhyde plus 0.2% arginine 2 Neutral TF plus 100 p.p.m. acetaldehyde plus 0.05% glycine 2 Alkaline TF, control 7 Alkaline TF 1 plus 100 p.p.m. acetaldehyde l4 Alkaline TF 1 plus 100 p.p.m. acetaldehyde plus 0.05% lysine 4 Alkaline TF 1 plus 100 p.p.m. acetaldehyde plus 0.2% arginine 4 Alkaline TF 1 plus 100 p.p.m. acetaldehyde plus 0.05% glycine 4 1 The alkaline tubing fluid contains 0.5% by weight diethy lamin'o ethanol and has a pH of 9.5.

The data of the above table demonstrates clearly that acetaldehyde causes a dramatic increase of digestion resistance and that this effect may be prevented by the pr ence in the tubing fluid of an amino acid. The data @1150 indicates that an alkaline tubing fluid has a stronger tanning effect than a neutral tubing fluid.

A further visual corroboration of the beneficial effect of the amino acids present in the tubing fluid is that the previously described orange-brown discoloration of the plain catgut is less marked, or absent in the samples that contain an amino acid. However, minute amounts of a brown pigment may be seen dissolved in the tubing fluid, which is believed to be due to a reaction between acetaldehyde and the added amino acid. When strips of white paper are added to the packaged catgut suture units as d scribed above the paper is stained brown in the presence of dissolved amino acid and acetaldehyde, whereas, the paper stays white when no amino acid is added.

EXAMPLE III The experiment described in Example II above is repeated substituting 0.05 cysteine, 0.2% proline, 0.01% aspartic acid, and 0.025 glutamic acid for the lysin arginine and glycine used in the alkaline tubing fluid of that example. The trypsin test indicates that the amino acids proline and cysteine are similar to lysine, arginine, and glycine in preventing tanning of the suture during storage by aldehydes present in the tubing fluid. The dicarboxylic amino acids, aspartic and glutamic acid are not effective at the concentrations indicated above.

4 EXAMPLE 1v Samples of plain catgut, size 2-0, are impregnated with an amino acid by immersion for ten minutes in dilute aqueous alcohol solution of glycine, proline, lysine and arginine. The sutures so treated are stored in foil packages containing tubing fluid and 100 p.p.m. of acetaldehyde. The effect of the amino acid present in the suture on tanning during storage is similar to the effect obtained by dissolving the amino acid in the tubing fluid.

EXAMPLE V Ten strands of plain catgut and ten strands of chromic catgut are sealed in individual packets with tubing fluid having the composition indicated in Table 2 below. This table also indicates the digestion time (average of ten samples) after five weeks aging. The digestion time was determined by incubation of the catgut samples in a 2% trypsin solution as described in Example I above.

This experiment shows that 0.025% glycine is sufiicient to produce the desired eflect. However, the concentration of amino acid may be adapted to the specific amino acid and tubing fluid used. The solubility of glycine increases in alcohol-water mixture with increasing water content, being 0.1% in isopropanol and 0.15% in 80% isopropanol. The preferred concentration of glycine in a isopropanol tubing fluid is 0.04% as this amount of glycine is below the solubility limit and yet is still sufficient to be effective.

EXAMPLE VI Three dilferent tests may be used to demonstrate the eflect of an amino acid-containing tubing fluid on the tanning of collagen sutures during storage.

Break time test In this test, catgut suture pieces of about 8 inches in length are formed into a loop and suspended under a constant load while immersed in a solution of the activated proteolytic enzyme papain. This papain solution is kept at a temperature of F. A mechanism is provided which automatically registers the time period, expressed in hours, required for the immersed catgut suture to be digested to such a degree that it breaks under the constant load. The apparatus is similar to that described by K. L. Lion and I. W. Sizer, Archives of Surgery, 48, page 120, 1944. From three to six tests are run on each sample, and the average value is reported as the break time. The more highly tanned and more resistant to the digestion the collagen suture is, the longer the break time.

Weight loss test weight is called the weight loss." The incubation temperature and incubation time are 45 C. and 45 minutes, respectively, for plain catgut, and 50 C. and 60 minutes from chromic catgut. Two tests are run on each sample, and the average value is reported as the weight loss. The more highly tanned and more resistant the collagen suture is to digestion, the smaller the weight loss.

Shrink temperature The shrink temperature is a commonly accepted measure of the degree of tanning or of the hydrothermal stability of collagen. It is the temperature at which collagen contracts or shrinks when gradually heated while it is kept immersed in water. The value given is the average of three tests on each sample. The more highly tanned and the more resistant the collagen suture is to absorption, the higher the shrink temperature.

Catgut strands, 12 inches to 60 inches in length, are heated for one hour at 137 C. and inserted in aluminum foil packets. Two and one-half milliliters of the tubing fluid, identified in Table 3 below, is added to each packet. The alkaine tubing fluid contains 0.05% by weight diethylamino ethanol. The packets are sealed hermetically and sterilized by irradiation with a linear accelerator type electron beam machine, the dosage being 2.5 megarads. After storage for ten weeks the samples are subjected to the tests described above in this example. The results summarized in Table 3 show that, within the range of variability that is to be expected in any type of biological or biochemical testing, the presence of glycine results in reduced tanning (lower digestion resistance).

TABLE 3 Catgut Sample-Type and Tubing Break Weight Shrink Fluid (TF) Time, Loss, Temp.,

hours percent 0.

Plain, 6-0, Sheep Intestine:

Alkaline NF+ Glycine" 0. 5 84 56 Alkaline TF 1. 64 60 Chromic, 6-0, Sheep Intestin Alkaline TF+ Glycine* 1. 8 86 61 Alkaline TF 2. 4 60 63 Plain, 3-0, Sheep Intestine:

Neutral TF+ Glycine* 2. 7 52 54 Neutral TB 4. 3 45 57. 5 3. 6 38 57. 5 5. 7 36 62 6.6 34 61 8. 8 38 61. 5 Plain, 3-0, Beef Intestine:

Alkaline TF-l- Glyeine* 5. 6 58 Neutral TF 7. 5 61 Ohromic, 3-0, Beet Intestine:

Alkaline TF-l-Glycine* 7. 5 54 62 Neutral TF 14.0 23 72 *0.04% Glycine.

EXAMPLE VII Catgut suture samples are prepared in the same manner as described in Example VI above. However, to the Tubing Fluid of a number of samples was added 37 p.p.m. of formaldehyde. The sealed and irradiated samples are tested after 3 and 6 months aging. The results for both table.

TABLE 4 Digestion Catgut Samples Ty%e and by Trypsin, Shrink Tubing Fluid (T Time in Temp., C.

. Days Plain, 6-0, Sheep Intestine:

Alkaline TF, Control 1 62. 5 Alkaline TF+37 p.p.rn. Formaldehyde 4 68 Alkaline TF+37 p.

hyde+ Glycine" 2 63. 5 Chrornic, 6-0, Sheep Intestine:

Alkaline TF, Control 2 64 Alkaline TF+37 p.p.m. Formaldehyde 6 68. 5 Alkaline TF+37 p.p.m. Formaldehyde+ Glycine* 3 65. 5 Plain, 2-0, Sheep Intestine:

Alkaline TF, Control 1 55. 5 Alkaline TF+37 p.p.m. Formaldehyde 2 58 Alkaline TF+37 p.p.m. Formalde hyde+ Glycine" 1 57. 5 Chromic, 2-0, Sheep Intestine Alkaline TI", Control. 2 64 Alkaline TF+37 p.p.m Formaldehyde 3 68. 5 Alkaline TF+37 p.p.m. Formaldehyde+ Glycine* 2 64 *0.04% Glycine.

What is claimed is:

1. A tubing fluid for absorbable sutures consisting essentially of a solution containing at least 0.01 percent by Weight of a monocarboxylic amino acid in a solvent comprising from 75 percent to 96 percent by weight of an alcohol selected fro mthe group consisting of ethanol, propanol, and isopropanol and from 4 percent to 25 percent by weight of water.

2. A tubing fluid for absorbable sutures consisting essentially of a solution containing at least 0.01 percent by weight of a monocarboxylic amino acid salt in a solvent comprising from 75 percent to 96 percent by weight of an alcohol selected from the group consisting of ethanol, propanol, and isopropanol and from 4 percent to 25 percent by weight of water.

3. The tubing fluid of claim 1 wherein the alcohol is isopropanol.

4-. The tubing fluid of claim 2 wherein the alcohol is isopropanol.

5. The tubing fluid of claim 1 wherein the alcohol is ethanol.

' 6. The tubing fluid of claim 2 wherein the alcohol is ethanol.

7. The tubing fluid of claim 1 wherein the monocarboxylic amino acid is arginine.

8. The tubing fluid of claim 1 wherein the mono carboxylic amino acid is lysine.

9. The tubing fluid of claim 1 wherein the monocarboxylic amino acid is glycine.

10. The tubing fluid of claim 1 wherein the monocarboxylic amino acid is proline.

11. The tubing fluid of claim 1 wherein the monocarboxylic amino acid is cysteine.

12. A tubing fluid for absorbable sutures consisting essentially of a solution containing at least 0.01 percent by weight of a monocarboxylic amino acid in a solvent comprising percent by weight isopropyl alcohol and about 10 percent by weight water.

13. The tubing fluid of claim 12 wherein the monocarboxylic amino acid is arginine.

14. The tubing fluid of claim 12 wherein the monocarboxylic amino acid is lysine.

15. The tubing fluid of claim 12 wherein the monocarboxylic amino acid is glycine.

8 References Cited UNITED STATES PATENTS 2,000,152 5/1935 Walker 252403 5 2,094,367 9/1937 Missbach 252403 X 2,518,233 8/1950 Hall 252403 X LEON D. ROSDOL, Primary Examiner.

M. WEINBLA'IT, Assistant Examiner. 

1. A TUBING FLUID FOR ABSORBABLE SUTURES CONSISTING ESSENTIALLY OF A SOLUTION CONTAINING AT LEAST 0.01 PERCENT BY WEIGHT OF A MONOCARBOXYLIC AMINO ACID IN A SOLVENT COMPRISING FROM 75 PERCENT TO 96 PERCNET BY WEIGHT OF AN ALCOHOL SELECTED FROM THE GROUP CONSISTING OF ETHANOL, PROPANOL, AND ISOPROPANOL AND FROM 4 PERCENT TO 25 PERCENT BY WEIGHT OF WATER. 